Drug discovery is a costly affair where one of the major expenses in terms of money and time is the in vivo studies. In order to reduce these costs a large number of in vitro models are developed and applied as filters to select the most suitable compounds for the in vivo studies. However, in vitro models are often too simplified and may as such be misleading in the decision-making process. Hence, there is a demand for intermediate models that are more reliable than in vitro models and at the same time faster and cheaper than traditional vertebrate in vivo models. Insects may serve this function and fruit flies are currently used as intermediate pharmacodynamic (PD) models by EnVivo Pharmaceuticals Inc., which develops CNS drugs.
There are various problems with existing in vitro testing. It is impossible to run in vitro assays to account for all biological events that occur in vivo. There are biological events that are not yet understood or shortcomings in the existing in vitro assays, e.g. assays may lack important features that are present in vivo, including active transporter molecules, metabolic enzymes, or even unforeseen biological events. Despite the obvious shortcomings, the in vitro models are heavily used in the drug discovery process where most pharmaceutical companies use large batteries of in vitro screens.
Testing compounds in a large number of in vitro assays may not always reflect the in vivo behavior. In fact, it is not unusual that compounds that have acceptable in vitro profiles turn out to have inadequate in vivo profiles. On the contrary, compounds may be discarded for wrong reasons. Thus, there is a requirement for intermediate in vitro/in vivo models, which could support the drug discovery research with improved data and hereby reduce the number of expensive in vivo experiments.
In vitro models are used with the supposition that each of the models reflects one single and isolated in vivo biological event. However, the large number of in vitro models that are used in the discovery phase (Ruiz-Garcia et al. 2007) are aimed at reflecting the complexity of the in vivo biology where numerous biological events take place in multiple compartments. A major limitation by using many in vitro models is the lack of interplay between different biological events and the interplay between different compartments. However, one major advantage by using insects as intermediate models is that these models fulfill the requirement of a complex interplay not only between different components of the brain barrier structure but also between the different compartments that appear in the insects since they are living species with compartments that to a large extent are similar to vertebrate.
The vertebrate blood-brain barrier (BBB) represents the physiological barrier between the brain tissue and blood vessels, which restricts the exchange of solutes and regulates absorption of exogenic agents (e.g. drugs) from the blood into the brain. The function of the central nervous system (CNS) requires a highly regulated extra-cellular environment. Anatomically the BBB in vertebrates is comprised of microvascular endothelia cells interconnected via highly specialized tight junctions (TJs), which provide a diffusion barrier and thus play a central role for permeability. Recently identified components of TJs include the claudins, a family of four-transmembrane-span proteins that are suggested to be responsible for the barrier-function of TJs (Turksen and Troy 2004). Penetration of BBB is one of the major hurdles in the development of successful CNS drugs. On the other hand, when penetration of the BBB occurs it may cause unwanted side effects for peripheral acting drugs (Schinkel 1999) (for review see Pard ridge 2002).
BBB penetration is usually classified as chemistry- or biology-based. The chemistry-based penetration is linked to the lipid mediated passive diffusion, which depends on physiochemical properties of the molecule, i.e. small hydrophobic molecules tend to penetrate the BBB more readily than large and hydrophilic molecules. The biology-based penetration involves compounds that are substrates for the endogenous BBB influx or efflux transport systems, e.g. many small molecules (e.g. drugs) have shown to be substrates for the P-glycoprotein (P-gp) transporter. The P-gp's are transporter proteins located in the walls of the cells that make up the BBB (Schinkel 1999) and they are conserved among taxa as diverse as protozoa, plants, insects and mammals (in Gaertner et. al. 1998). P-gp's are present in many cell-types and they play important roles in drug absorption, disposition, metabolism, and toxicity (Xia et al. 2006).
Obviously, it is crucial to have an understanding of the BBB penetration in drug discovery projects and preferably, this should be obtained without using excessive number of in vivo studies. Consequently, several in vitro BBB absorption models are developed to predict the in vivo behaviour of test compounds. However, even complex in vitro models which include the P-gp transporter systems (Di and Kerns 2003, Summerfield et al. 2005) seem not to meet the intricate complexity of the TJs and therefore may not describe the in vivo behavior very well. This is strongly indicated in an extensive BBB absorption study, in which 22 compounds were tested in ten different in vitro BBB absorption models (Garberg 2005). None of the ten models showed any correlation between in vitro and in vivo permeability. This indicates that specific BBB models not necessarily provide better prediction than non-BBB derived models. Furthermore, it was suggested that protein binding, blood-flow, metabolic stability and lipophilicity, as well as affinity for other transporters in the BBB are factors needed to be considered when predictions of in vivo brain distribution is to be made. Consequently, it seems as in vitro models are mainly suited for qualitative measurements of compounds that penetrates BBB by passive diffusion or compounds that undergo efflux via the P-gp transporter (Garberg 2005).
Certain invertebrates have served as useful models for understanding many different biological processes. Especially the fruit fly, Drosophila melanogaster is a well-recognized model research organism, which have made significant contributions to the understanding of genetics, neurobiology, molecular biology etc. (Gullan and Cranston 2000). Generally, insects and vertebrates have many physiological features in common. They are multi cell organisms with complex compartmentalized nervous systems for specialized functions like vision, olfaction, learning, and memory. The nervous systems of the insects respond physiologically in similar ways as in vertebrates with many identical neurohormones and receptors. Insects have avascular nervous systems in which hemolymph bathes all outer surfaces of ganglia and nerves. Therefore, many insects require a sophisticated BBB system to protect their CNS from plant-derived neurotoxins and to maintain an appropriate ionic microenvironment of the neurons. In fact, also in insects a sophisticated BBB system has been an evolutionary advantage. In insects this BBB is mainly based on the glia cell system which certainly shifted to the endothelial system as a response to the increased importance of the microvasculature in the vertebrate brain. In support of this view is the appearance of the glia system in elasmobranch fish and the remnants of the glia barrier in modern mammalian CNS. Thus insects possess a BBB which is an important component in the ensheathment of the nervous system. The BBB:s in insects are highly sophisticated but varies in structure between different insect orders. Thus insects with highly sophisticated brain barriers with complex integrative components that mimic the vertebrate barriers will be excellent models for documentation of penetration of various molecules through this structure.
US20050132425A1 discloses a transgenic fly that expresses the Italian mutant version of the human Abeta42 peptide of human amyloid-beta precursor protein (APP), and a double transgenic fly that expresses both the Tau protein and the human Abeta42Italian peptide of human amyloid-beta precursor protein (APP). The transgenic flies provide for models of neurodegenerative disorders, such as Alzheimer's disease. US20050132425A1 further discloses methods for identifying genetic modifiers, as well as screening methods to identify therapeutic compounds to treat neurodegenerative disorders using the transgenic flies.
WO04006854A2 discloses a method for screening for the effect of a test agent on a population of insects comprising the steps of providing a population of specimen, administering at least one test agent to the population, creating a digitized movie showing the movements of the insects, measuring at least one trait of the insects of the population with the effect of the test agent. The document also provides a method for preparing a medicament useful for the treatment of a mammalian disease.
Marsh and Thompson (Marsh and Thompson 2006) teach that insects are very useful as model systems due to simplicity combined with fast reproducibility. Some of the models (with Drosophila) have demonstrated their efficiency for testing relevant drugs and revealed concordance of drug efficacy in flies and mammals for diseases like Huntingtons's, Parkinson's and Alzheimer's.
Marsh and Thompson (Marsh and Thompson 2004) suggest that the dominant neurodegenerative diseases of man can be faithfully modelled in insects, such as Drosophila (fruitfly), since they exhibit the key features of these diseases such as slowly progressing degeneration, late onset, formation of abnormal protein aggregates etc. According to the authors the ability to manipulate such engineered organisms allows pathogenic mechanisms to be identified and potential pharmacologic regimens to be rapidly tested. The authors suggest that the excellent agreement to date of pharmacologic treatments that are effective in suppressing pathology in both flies and in mice gives growing confidence that invertebrate model organisms can productively speed the identification of agents that are likely to be effective at treating diseases in mammals.
As appears from the above mentioned literature the prior art is primarily directed to the testing of compounds in flies (Drosophila) for use in the treatment of human neurodegenerative diseases. However, there is still a need to identify appropriate screening models beyond the commonly used in vitro testing methods to determine/assess blood-brain barrier penetration of drugs. In this regard it should be borne in mind that flies have septate junctions and not tight junctions like vertebrate and locust, moth and cockroaches.
There is an urgent need for more sophisticated screening models in drug discovery but also in testing of CNS toxicity of chemicals on market with less known effects on brain function.
Thus, in drug discovery there is:    a) a need for efficient screening of compounds aimed at targets within the CNS system. This screening is preferentially performed in insect models with intact BBB function and will contribute to a positive selection of compounds penetrating the BBB. Such screening comprises low molecular weight compounds within a number of indications (e.g. pain, epilepsy, Parkinson, schizophrenia, Alzheimer, sleep disorders, anxiety, depression, eating disorders, drug abuse including smoking).    b) a need for efficient screening of compound which are targeted for efficacy outside CNS and when penetrating CNS may induce non acceptable side effects.    c) a need for efficient screening in insect models characterized by selective changes in the function of the BBB. Such screening comprises low to very high molecular weight compounds or peptides or macromolecules in diseases characterized by deteriorated BBB function (e.g. ischemic stroke, traumatic brain injury, drug abuse, neurodegenerative diseases like Parkinson and Alzheimer, epilepsy, infections, inflammation like meningitis and MS, HIV).
There is also a need for screening of chemical compounds on the market, which has not been classified or documented for there potential neurotoxicity.